USE OF THE U94 MOLECULE OF HUMAN HERPESVIRUS 6 AND DERIVATIVES THEREOF TO INCREASE OR INDUCE THE EXPRESSION OF THE HLA-G MOLECULE

Abstract

A method of enhancing or inducing the expression of the HLA-G molecule by human cells is provided. The method includes administering the human Herpesvirus 6 U94 molecule or a derivative thereof, or a gene expression vector including and expressing the human Herpesvirus 6 U94 gene to a patient in need thereof or to an in vitro cell, tissue, or organ culture.

Claims

1-10. (canceled)

11. A method of administering an immuno-suppression and/or anti-inflammatory treatment to a subject in need thereof, the method comprising administering to the subject an amount of human Herpesvirus 6 U94 molecule or derivative thereof, the amount being effective in enhancing or inducing HLA-G expression in cells and/or tissues of the subject.

12. The method of claim 11, wherein the subject is a human being.

13. The method of claim 12, wherein the subject suffers from an autoimmune disease, a tumour disease, or infertility.

14. The method of claim 11, wherein the human Herpesvirus 6 U94 molecule or derivative thereof is administered as an adjuvant in a therapeutic immunosuppressive treatment in a bone marrow transplant, a stem cell transplant, or a solid organ transplant.

15. The method of claim 14, wherein the solid organ transplant is a kidney, liver, heart, lung, pancreas, or intestine transplant.

16. The method of claim 11, wherein the human Herpesvirus 6 U94 molecule or derivative thereof is co-administered with an immunosuppressive drug.

17. A method of administering an immuno-suppression and/or anti-inflammatory treatment to a subject in need thereof, the method comprising administering to the subject an amount of a gene expression vector including and expressing the human Herpesvirus 6 U94 gene, the amount being effective in enhancing or inducing HLA-G expression in cells and/or tissues of the subject.

18. An in vitro method for modulating the expression of the HLA-G molecule in a mammalian cell, tissue, or organ culture, the in vitro method comprising administering to said culture the human Herpesvirus 6 U94 molecule or a derivative thereof.

19. The in vitro method of claim 18, comprising administering to the mammalian cell, tissue, or organ culture an amount of the human Herpesvirus 6 U94 molecule or derivative thereof which is effective in enhancing or inducing HLA-G expression.

20. An in vitro method for modulating the expression of the HLA-G molecule in a mammalian cell, tissue, or organ culture, the in vitro method comprising administering to said culture a gene expression vector including and expressing the human Herpesvirus 6 U94 gene.

21. The in vitro method of claim 20, wherein the gene expression vector is a herpes amplicon vector.

Description

DESCRIPTION OF THE INVENTION

[0027] Despite the importance of the HLA-G molecule in regulating the immune response has been extensively studied and demonstrated, many problems remain unsolved for the use of this molecule in the therapeutic field.

[0028] The first problem is due to the trimolecular nature of HLA-G, formed by the heavy chain, beta 2 microglobulin and the peptide. In addition to being difficult to manufacture, this structure has low stability.

[0029] Moreover, the data available today is based on synthetic molecules or on molecules purified from cell line cultures transfected with the HLA-G gene. Interaction between HLA-G molecules (multimers) and association with other molecules are therefore not taken into consideration. For this reason, the development of a therapy-compatible HLA-G molecule is still progressing slowly.

[0030] The object of the present invention is to overcome these and other drawbacks of the prior art.

[0031] To this end, a first aspect of the present invention is the human Herpesvirus 6 U94 molecule or a derivative thereof, for use in a therapeutic immuno-suppression and/or anti-inflammatory treatment method, said method comprising administering to a subject an amount of U94 or derivatives thereof which is effective in modulating, i.e. enhancing or inducing HLA-G expression in said subject. The subject is preferably a mammal, more preferably a human being.

[0032] In the scope of the present description, the term “modulating” is sometimes used with reference to HLA-G expression in mammalian cells and/or tissues. It should be emphasized that, in this context, this term has the meaning of “enhancing or inducing”. The same applies to the variant “modulation”, which has the meaning of “enhancement or induction”.

[0033] The present invention is extremely advantageous compared to the state of the art, since an HLA-G molecule directly produced by the cells/tissues of the organism to be treated greatly facilitates the use of this protein for therapeutic purposes. In this way, synthesis and extraction problems are avoided and a molecule fully compatible with the parent cells/tissue is obtained. Moreover, thanks to the invention, the HLA-G molecules are induced both as membrane proteins and in the soluble form, with the advantages of both a local and a systemic action.

[0034] The HLA-G molecule produced by means of the present invention was shown to have a tolerogenic function, by inhibiting the cytotoxic activity of NK cells and cytotoxic T lymphocytes, the allogeneic T lymphocyte response, dendritic cell maturation, and by inducing the maturation of regulatory T cells and IL-10-expressing dendritic cells.

[0035] The modulation of the HLA-G molecule according to the invention can be used together with drugs capable of regulating the immuno-inhibitory response. This benefits the patient by reducing the required immunosuppressive drug levels and those required to induce better tolerance.

[0036] Accordingly, a second aspect of the invention is the human Herpesvirus 6 U94 molecule or a derivative thereof, for the above indicated use, as an adjuvant, in the therapeutic immunosuppressive treatment, co-administered with an immunosuppressive drug such as, but not limited to, FK506, ciclosporin, cortisone, mycophenolate, azathioprine, or everolimus.

[0037] Pharmaceutical preparations containing the active ingredients combined with suitable pharmaceutically acceptable excipients and/or carriers are conveniently used for administering the U94 molecule and derivatives thereof. The concentration of the active ingredients must be sufficient to obtain the modulation of the HLA-G molecule with an immuno-inhibiting effect. The selection of excipients and/or carriers to be used in the pharmaceutical preparations of the invention is within the skills of the person skilled in the art.

[0038] In the scope of the present invention, the human Herpesvirus 6 U94 molecule can be administered as a protein or derivatives thereof, but also in gene therapy, by using a gene expression vector including and expressing the human Herpesvirus 6 U94 gene.

[0039] Therefore, a third aspect of the present invention is a gene expression vector including and expressing the human Herpesvirus 6 U94 gene, for the aforementioned therapeutic uses of the invention.

[0040] According to a preferred embodiment, the gene expression vector is a herpes amplicon, however any gene expression vector suitable for use in gene therapy, particularly in humans, can be used in the implementation of the present invention.

[0041] It should be specified that both the human Herpesvirus 6 U94 protein and its encoding gene are known per se and that their sequences belong to the state of the art. In this regard, there can be mentioned, by way of example, the UniProtKB database (entries Q9WSZ6 and Q00683), and the papers by Gompels U A. et al., Virology, 1995 May 10; 209(1):29-51 and Mirandola P. et al., J Virol. 1998; 72:3837-3844.

[0042] The functional and/or therapeutic activity of the doses of active ingredients (proteins or nucleic acids) can be evaluated in vitro by various methods that measure the induction of HLA-G. For example, the immuno-modulating function of the HLA-G molecule expressed upon treatment with U94 or derivatives thereof or with the gene expression vector expressing the U94 gene can be evaluated in vitro through different systems that include, but are not limited to, the inhibition of the activation of NK cell lines against cell targets lacking HLA class I expression, such as for example the K562 cell line. The therapeutic dosage for the modulation of the expression of the HLA-G molecule can be extrapolated in vitro by using the U94 molecule or a derivative thereof or the gene expression vector expressing the U94 gene on cell lines representing the cell target of the action. The dosage also depends on the mode of administration. For example, in some applications, such as the treatment of bone marrow transplants, the cells in culture are treated with soluble suspensions of the active ingredients. The dosage to be used refers to the correct amount of the U94 molecule or a derivative or vector thereof that is able to cause enhancement/induction of expression of the HLA-G molecule in sufficient quantity to perform its immuno-inhibitory function. The therapeutic effect may include, but is, not limited to, the inhibition of the cytotoxicity of NK cells and T lymphocytes, the allogeneic T lymphocyte response, dendritic cell maturation, and the induction of regulatory T cells and IL-10-expressing dendritic cells. The precise dosage will depend on the number of cells to be treated, the size of the tissue and the nature of the same. The person skilled in the art is able to determine the correct dosage by routine experimentation.

[0043] Diseases and disorders or conditions associated with the need to induce an immune-tolerant state that can be treated according to the present invention include, but are not limited to, bone marrow transplantation, stem cell transplantation, transplantation of solid organs (such as for example kidney, liver, heart, lung, pancreas, intestine); autoimmune diseases (such as for example rheumatoid arthritis; multiple sclerosis), tumours, infertility.

[0044] The invention falls within the scope of immunosuppressive therapies to be implemented in the clinical setting. For this purpose, the U94 molecule or derivatives thereof or the gene expression vector expressing the U94 gene can be provided in the form of a kit or a pharmaceutical composition, with which a package leaflet can optionally be associated in the form regulated by law for the production, use and sale of pharmaceutical compounds, whose notes will include approval, use and sale for use in mammalian cells/tissues.

[0045] A further aspect of the present invention is an in vitro method for enhancing or inducing the expression of the HLA-G molecule in mammalian cell, tissue or organ cultures, which comprises administering to said cell, tissue or organ cultures the human Herpesvirus 6 U94 molecule or a derivative thereof, or a gene expression vector, preferably a herpes amplicon vector, expressing the human Herpesvirus 6 U94 gene, in an amount suitable to enhance or induce HLA-G expression in the treated cultures.

[0046] In one embodiment of the invention, the treated cell cultures are primary tumour-derived cells.

[0047] The experimental section that follows is provided for illustration purposes only and does not limit the scope of the invention as defined in the appended claims.

Experimental Section

U94 Production

[0048] M15 bacterial cells transformed with the pQE-rep plasmid were grown at 37° C. in LB containing 100 μg/ml ampicillin and 50 μg/ml kanamycin up to OD660 nm=0.4-0.6 and subsequently induced with 2 mM IPTG. After 1, 3 and 5 hours of induction at 37° C., the samples were collected by centrifugation and analysed for the presence of recombinant proteins. Cell pellets from 500 ml culture were suspended in 50 ml of lysis buffer (50 mM Tris-HCl pH 8, 2 mM EDTA, 1 mM DTT, 1 μg/ml aprotinin, 1 mM PMSF, 100 μg/ml lysozyme and 1% Triton X-100). After incubation for 30 minutes at room temperature, the suspension was cleared by sonication (three cycles of 15 s with a 15 s interval) and centrifuged for 15 minutes at 13,000 rpm at 4° C. Samples from supernatant (soluble fraction), pellet (insoluble fraction) and total lysate were analysed by SDS-PAGE. U94/REP protein was recovered mostly from the insoluble fraction, then the samples were solubilized in lysis buffer containing increasing molar concentrations of urea (2, 4, 6, 8 M). Each fraction was analysed by SDS-PAGE and Western blot. Complete solubilization of the protein was achieved with lysis buffer supplemented with 6 M urea. The fraction containing the recombinant protein was dialyzed to remove Triton X-100 residues and purified under denaturing conditions by hydroxyapatite chromatography, using the lysis buffer in a phosphate gradient. The sample was applied to the column after equilibration with lysis buffer without Triton, pH 6.8. The resin was washed with a 10-200 mM sodium phosphate buffer, and the protein was eluted with 500 mM phosphate. Each fraction was examined by spectroscopy at 280 nm and subsequently analysed by SDS-PAGE and Western blot. Endotoxin levels were tested and were below 0.5 EU/μg protein in all assays performed.

HLA-G Induction

[0049] HUVEC cells were treated with 3 μg/ml recombinant U94/REP protein. Induction of HLA-G expression was determined by flow cytometry. Staining was performed with anti-HLA-G moAb (87 G moAb) (Exbio, Praha, Czech Republic), and isotypic controls (Exbio, Praha, Czech Republic). Analysis was carried out with a FACSCanto II cytometer and the FlowJo software. Results were expressed as MFI (mean fluorescence intensity).

In Vitro Studies

Cytotoxicity (MTT Assay)

[0050] 100 μl of U937 cells at a density of 1×10.sup.6/m1 were plated and treated with U94 (3 μg/ml) for 24 hours. Subsequently, 10 μl of MTT (Sigma-Aldrich, Milan, Italy) were added for 4 hours at 37° C. The cells were lysed by adding 100 μl of MTT solvent. The plates were read at 570 nm using the plate reader (Victor, PerkinElmer, Waltham, USA).

Immuno-Suppressive Activity of Treatment-Induced Molecules

[0051] Supernatants of HUVEC cell cultures treated or not treated with U94 were added to NK cell cultures (cell line NK92, ATCC-CRL2407), in a 1:5 ratio with the K562 target cells (ATCC-CCL243), characterized by the absence of HLA class I expression. NK cell activation status was determined by flow cytometry, by analysing CD107a expression. Briefly, the CD107a degranulation test was performed by incubating the cells for 3 hours at 37° C. with the Golgi Stop solution (Becton Dickinson) and subsequently by labelling with the anti-CD107a antibody (Becton Dickinson).

HLA-G Induction by U94

[0052] Human MDA-MB 231 mammary carcinoma cells in which U94 expression was induced by an HSV-1-based amplicon were analysed by ELISA test for expression of soluble HLA-G secreted in the culture supernatants and by immunohistochemistry for expression of membrane-bound HLA-G.

[0053] The differences between the groups were assessed by the Mann Whitney U test. A p value <0.05 was regarded as a statistically significant value.

Results

In Vitro Studies

U94-Induced HLA-G Expression

[0054] Cytotoxicity assessment has shown that the dose of U94 to be used is 3 μg/ml, as it induces the highest production of HLA-G with the lowest cellular toxicity (Table 1).

TABLE-US-00001 TABLE 1 Cytotoxicity analysis of the U94 protein U94 HLA-G; % cells Cytotoxicity; %  3 μg/ml 34.0 ± 2.5  5.2 ± 1.2 13 μg/ml 25.2 ± 3.6 29.0 ± 4.6 20 μg/ml  23. ± 4.1 49.0 ± 3.2

[0055] In the experiment shown in FIG. 1, HUVEC cells were treated with U94 (3 μg/ml). HLA-G expression has been shown as A, B) mRNA and C) membrane protein and D, E, F, G)) soluble. Following exposure to U94, HUVEC cells show increased expression of both the membrane-bound and the soluble form of HLA-G. In particular, after 24-48 hours from U94 transfection or addition of recombinant U94 to the culture medium of HUVEC cells there is an increase in the soluble form of HLA-G (HLA-G5/6) (FIG. 1 D, E, F, G).

[0056] U94 activates the HLA-G gene promoter. In the pGL3-Basic reporter plasmid (Promega, Madison, Wis.) a PCR-generated fragment containing the HLA-G promoter sequence followed by the luciferase gene was cloned. U94 activates the HLA-G promoter. FIG. 2 shows the results of this experiment, in which HUVEC cells (10{circumflex over ( )}6 cells) were co-transfected with 0.5 μg p94 plasmid encoding the U94 viral gene or with the corresponding empty vector (CTR), together with 0.5 μg of pGL3-HLA-G1500 (pHLA-G) or pGL3-B250 (p250) and 0.2 μg of pRL-Renilla luciferase control reporter vector. Luciferase expression was assessed after 48 hours. The results are expressed as mean fold activation values±SD in duplicate samples from two independent experiments.

[0057] Supernatants of HUVECs treated or not treated with U94 were added to NK cell cultures (NK92 cell line), in the presence of K562 target cells. Activation status was analysed by flow cytometry (CD107a). Supernatants of U94-treated HUVEC cells containing HLA-G in its soluble form reduce the activation status of NK cells (FIG. 3). In contrast, supernatants of untreated HUVEC cells do not interfere with NK cell activation status. These results demonstrate the tolerogenic effect of U94-induced HLA-G molecules.

[0058] The ability of HUVEC cells to form capillaries is increased by transfection of the same cells with U94 or HLA-G, or by the addition of recombinant U94 or HLA-G proteins to the cell supernatants (FIG. 4). The addition of a specific antibody against HLA-G blocks the formation of capillaries in HUVEC cultures transfected with U94 or HLA-G or added with recombinant U94 or HLA-G (FIG. 4). This result confirms that induction of angiogenesis by U94 is mediated by the HLA-G molecule. In the experiment in FIG. 4, HUVEC cells were transfected with plasmids coding for HHV-6 U94 gene (pU94) or human HLA-G (pHLA-G) or treated with the recombinant U94 or HLA-G proteins. All samples were also tested with a specific antibody against HLA-G (αHLA-G Ab, 7.5 ng/ml). Images were obtained after a 24-hour incubation.

[0059] U94 expression in MDA-MB 231 cells induces HLA-G secretion in the supernatant (FIG. 5A). Membrane-bound HLA-G expression is evident in U94-expressing MDA-MB 231 cells both in a three-dimensional in vitro culture model (FIG. 5B) and in vivo in U94-expressing MDA-MB 231 cells inoculated in mouse (FIG. 5B). In particular, FIG. 5A shows the results of the analysis of soluble HLA-G levels in the culture supernatants of MDA-MB 231 cells in which U94 expression was induced by an HSV-1 based amplicon. NT: MDA-MB 231; EGFP: MDA-MB 231 with empty amplicon; U94: MDA-MB 231; EGFP: MDA-MB 231 with U94 amplicon. FIG. 5B shows the expression of membrane-bound HLA-G, as analysed by immunohistochemistry in U94-expressing MDA-MB 231 cells cultured in vitro in a three-dimensional system (left panel) or inoculated in mouse (right panel). Placental HLA-G expression was used as a positive control.